A major challenge for mammalian hosts is to maintain symbiotic relationships with the vast bacterial communities that colonize the intestines. The intestinal epithelium is the primary barrier between the intestinal microbiota and internal host tissues, yet little is known about how epithelial cells control bacterial interactions with the mucosal surface and limit microbial penetration of the intestinal barrier. Filling this knowledge gap is crucial, as many gastrointestinal diseases of humans originate with pathogenic invasions of the intestinal epithelium, or involve dysregulated interactions between the commensal microbiota and epithelial cells. In the previous project period, we showed that intestinal epithelil cell autophagy limits tissue invasion by opportunistically invasive commensals and the invasive intestinal pathogen, Salmonella typhimurium. We found that autophagy activation by S. typhimurium requires the innate immune signaling adaptor MyD88, and is regulated by interactions between the anti-autophagy factor BCL2 and the essential autophagy protein Beclin 1. Our initial findings thus identify autophagy as an important epithelial cell-autonomous mechanism of antibacterial defense and indicate that epithelial autophagy activation is tightly regulated. During the next project period, we propose to build upon these findings to gain a deeper understanding of how antibacterial autophagy is regulated in the intestinal epithelium, and to elucidate the physiological significance of epithelial autophagy for maintaining homeostasis with the microbiota. In Aim 1, we will use in vivo mouse models to further delineate the role of BCL2 in regulating antibacterial autophagy in the intestinal epithelium. In Aim 2, we will use in vivo approaches to determine how MyD88 regulates antibacterial autophagy in intestinal epithelial cells. In Aim 3, we will use mice harboring epithelial cell-specific autophag defects to test the hypothesis that autophagy promotes the clearance of opportunistically-invasive commensal bacteria. These studies will significantly advance our understanding of how intestinal epithelial cells promote homeostasis with the diverse bacterial community in the intestine. Further, our findings should suggest novel strategies for enhancing intestinal epithelia cell autophagy to protect against intestinal infections and inflammatory diseases.